Structural basis of binding the unique N-terminal domain of microtubule-associated protein 2c to proteins regulating kinases of signaling pathways

Isoforms of microtubule-associated protein 2 (MAP2) differ from their homolog Tau in the sequence and interactions of the N-terminal region. Binding of the N-terminal region of MAP2c (N-MAP2c) to the dimerization/docking domains of the regulatory subunit RIIα of cAMP-dependent protein kinase (RIIDD2) and to the Src-homology domain 2 (SH2) of growth factor receptor-bound protein 2 (Grb2) have been described long time ago. However, the structural features of the complexes remained unknown due to the disordered nature of MAP2. Here, we provide structural description of the complexes. We have solved solution structure of N-MAP2c in complex with RIIDD2, confirming formation of an amphiphilic α-helix of MAP2c upon binding, defining orientation of the α-helix in the complex and showing that its binding register differs from previous predictions. Using chemical shift mapping, we characterized the binding interface of SH2-Grb2 and rat MAP2c phosphorylated by the tyrosine kinase Fyn in their complex and proposed a model explaining differences between SH2-Grb2 complexes with rat MAP2c and phosphopeptides with a Grb2-specific sequence. The results provide the structural basis of a potential role of MAP2 in regulating cAMP-dependent phosphorylation cascade via interactions with RIIDD2 and Ras signaling pathway via interactions with SH2-Grb2.


Contents
Table S1.Alignment of selected vertebrate Grb2 SH2 binding sequences of EGFR and CD28.
Table S2.The CYANA running script CALC.cya and additional settings in init.cya           ).Abl kinases The spectra were measured for 20 hrs upon addition of the kinase.MAP2c specifically 15 N-labeled on tyrosines was used because1 H, 15 N HSQC spectrum of uniformly labeled MAP2c is not sufficiently resolved in the region of the tyrosine peaks.This approach allowed us to observe changes at all tyrosines directly, simultaneously, and in a real time.Peak heights of unphosphorylated Tyr50 (magenta), Tyr67 (red), Tyr252 (blue), Tyr265 (cyan), and Tyr325 (purple) are plotted as circles.Peak heights of phosphorylated pTyr67 (red) and of an unassigned pTyr phosphorylated by Abl (black) are plotted as crosses.The results confirm that Fyn phosphorylates selectively Tyr67 of MAP2c.

Figure S3 .
Figure S3.Comparison of structural models of the N-MAP2c:RIIDD 2 complex predicted by AlphaFold multimer and AlphaFold 3 (B), and of a model of the binding region of free MAP2c predicted by AlphaFold 2 with the representative structure calculated in our study.

Figure S4 .
Figure S4.Overlay of 1 H-15 N HSQC spectra of selectively [ 15 N-Tyr] labeled MAP2c before and after phosphorylation by Fyn and Abl, and changes in the tyrosine peak heights in the course of Fyn and Abl phosphorylation.

Figure S6 .
Figure S6.Snapshots of molecular dynamics simulations of phosphopeptides containing the pTyr-Val-Asn-Val sequence and the pTyr-Ser-Asp-Thr sequence in complex with Grb2-SH2 and distances between phosphopeptide pTyr and Arg86 of Grb2-SH2.

Figure S7 .
Figure S7.Results of ITC analysis of Fyn-phosphorylated MAP2c titrated by RIIα-PKA in absence and presence of 100 μM Grb2.

Figure S1 .
Figure S1.Results of ITC analysis of MAP2c titrated by RIIα-PKA (A) and RIIDD 2 (B).The concentration of MAP2c and N-MAP2c in the cell was 25 μM, the concentration of RIIα-PKA and RIIDD 2 in the syringe was 600 μM.Results of the fits in the insets document that stoichiometry and enthalpy of the binding was determined reproducibly, but the binding isotherms are too steep for reliable estimation of K D .

Figure S3 Figure
Figure S3Comparison of structural models of the N-MAP2c:RIIDD 2 complex predicted by AlphaFold multimer (A) and AlphaFold 3 (B), of a model of the binding region of free MAP2c predicted by AlphaFold 2 (C), and of the representative structure calculated in our study (D).The structures predicted by AlphaFold are colored (separately for N-MAP2c and RIIDD 2 ) according to the AlphaFold confidence score: blue, pLDDT>90, cyan, 90>pLDDT>70, yellow, 70>pLDDT>50, and red, pLDDT<50.In the experimental structure, N-MAP2c and RIIDD 2 are shown in magenta and gray, respectively.

Figure S6 .
Figure S6.Snapshots of molecular dynamics simulations of phosphopeptides containing the pTyr-Val-Asn-Val sequence (A) and the MAP2c pTyr-Ser-Asp-Thr sequence (B) in complex with Grb2-SH2.Ten snapshots of the phosphopeptide conformations, sampled each 5 ns, are displayed in gray.Only one Grb2-SH2 structure (gold) is shown for the sake of clarity.Residues Asp, Asn, and pTyr of the phosphopeptides are shown as bright green, purple, and magenta sticks, respectively.Nitrogen, oxygen, and hydrogen atoms of the Asn amide and Asp carboxy groups are displayed in blue, red, and white, respectively.Backbone atoms of Grb2-SH2 Lys109, forming a hydrogen bond with the phosphopeptide Asn in the pYXN motif, are displayed in the ball-and-stick representation.Distances between phosphopeptide pTyr and Arg86 of Grb2-SH2 are shown in blue for the the pTyr-Val-Asn-Val sequence (C) and in red for the pTyr-Ser-Asp-Thr sequence (D).

Figure S7 .
Figure S7.Results of ITC analysis of Fyn-phosphorylated MAP2c titrated by RIIα-PKA in absence (A) and presence of 100 μM Grb2 (B), and a control titration of 100 μM Grb2 by RIIα-PKA (C).The concentration of Fyn-phosphorylated MAP2c in the cell was 25 μM, the concentration of RIIα-PKA and RIIDD 2 in the syringe was 600 μM.The obtained binding isotherms in Panel A and B did not differ substantially.Considering the high Grb2 concentration, we can conclude that Grb2 does not interfere with RIIα-PKA binding.The isotherms were steep, resembling titration of unphosphorylated MAP2c by RIIα-PKA (Figure S1A) and did not allow us to estimate a quantitative value of K D .The control experiment (Panel C) showed that possible interactions between Grb2 and RIIα-PKA are too weak to influence the results.

Table S1 .
Alignment of selected vertebrate Grb2 SH2 binding sequences of EGFR and CD28.Tyrosines corresponding to human MAP2 Tyr67 are in bold, asparagines aligned with human MAP2 Asn69 are shown in green.

Table S2 .
The CYANA running script CALC.cya and additional settings in init.cya